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CN-119219783-B - Goat recombinant monoclonal antibody resisting rabbit IgG and preparation and application thereof

CN119219783BCN 119219783 BCN119219783 BCN 119219783BCN-119219783-B

Abstract

The application discloses a goat recombinant monoclonal antibody of anti-rabbit IgG, and preparation and application thereof, and belongs to the technical field of biology. The recombinant monoclonal antibody is prepared by taking purified rabbit serum IgG as an immunogen, immunizing a goat, obtaining a heavy chain variable region and a light chain variable region of the antibody through specific B cell screening and RT-PCR, respectively inserting variable region sequences into vector plasmids with constant region (heavy chain and light chain) genes, and purifying after transfection and expression. The recombinant monoclonal antibody provided by the application has high affinity with rabbit IgG, can recognize and detect rabbit IgG with high specificity and high sensitivity, can be used as a secondary antibody after being crosslinked by HRP, is applied to detection of rabbit IgG by methods such as Western immunoblotting, immunohistochemistry, ELISA and the like, is beneficial to obtaining more accurate detection and evaluation results, and reduces detection cost and interference of background signals.

Inventors

  • YING YU
  • HUO NA
  • WEI LI
  • ZHANG HONGFANG

Assignees

  • 杭州斯达特生物科技有限公司
  • 南京优宁维生物科技有限公司

Dates

Publication Date
20260512
Application Date
20241113

Claims (11)

  1. 1. The goat recombinant monoclonal antibody of the anti-rabbit IgG is characterized in that the amino acid sequence of a heavy chain variable region of the recombinant monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the recombinant monoclonal antibody is shown as SEQ ID NO. 2.
  2. 2. The goat recombinant monoclonal antibody of claim 1, wherein the heavy chain constant region of the recombinant monoclonal antibody has an amino acid sequence shown in SEQ ID NO.5 and the light chain constant region has an amino acid sequence shown in SEQ ID NO. 6.
  3. 3. A nucleic acid encoding the heavy chain variable region and the light chain variable region of the goat recombinant monoclonal antibody of anti-rabbit IgG of claim 1 or 2.
  4. 4. A nucleic acid according to claim 3, wherein said nucleic acid comprises: A nucleotide sequence shown as SEQ ID NO.3 for encoding a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 1; the nucleotide sequence shown as SEQ ID NO.4 is used for encoding a light chain variable region of the amino acid sequence shown as SEQ ID NO. 2.
  5. 5. A recombinant expression vector comprising the nucleic acid of claim 3 or 4.
  6. 6. A recombinant expression cell comprising the recombinant expression vector of claim 5, or the nucleic acid of claim 3 or 4.
  7. 7. Use of the nucleic acid of claim 3 or 4, or the recombinant expression vector of claim 5, or the recombinant expression cell of claim 6 for the preparation of goat recombinant monoclonal antibodies against rabbit IgG.
  8. 8. The method for preparing the goat recombinant monoclonal antibody of the anti-rabbit IgG of claim 1, which comprises the following steps: transfecting cells with the recombinant expression vector of claim 5 to obtain recombinant expression cells, culturing the transfected recombinant expression cells, collecting supernatant and purifying to obtain the anti-rabbit IgG recombinant monoclonal antibody; or culturing the recombinant expression cell of claim 6, collecting the supernatant and purifying to obtain the anti-rabbit IgG recombinant monoclonal antibody.
  9. 9. An HRP-labeled goat recombinant monoclonal antibody against rabbit IgG, wherein the recombinant monoclonal antibody is the goat recombinant monoclonal antibody against rabbit IgG of claim 1 or 2.
  10. 10. Use of the recombinant monoclonal antibody of claim 1 or 2 or 9, or the nucleic acid of claim 3 or 4, or the recombinant expression vector of claim 5, or the recombinant expression cell of claim 6, or the method of preparation of claim 8, for the preparation of a test rabbit IgG product.
  11. 11. The use according to claim 10, wherein the detection of rabbit IgG comprises any one or more of immunohistochemistry, western blotting or ELISA.

Description

Goat recombinant monoclonal antibody resisting rabbit IgG and preparation and application thereof Technical Field The application belongs to the technical field of biology, and particularly relates to a goat recombinant monoclonal antibody of anti-rabbit IgG, and preparation and application thereof. Background When people and animals develop diseases, the human and animals need to rely on autoimmune response, the specific immunity plays an important role, the humoral immunity occupies a certain position in the specific immunity, the humoral immunity mainly depends on various immunoglobulins, wherein IgG occupies about 75% -80%, and the human and animals are main antibodies of secondary immune response, and are also main target antibodies for serodiagnosis and detection after vaccine immunity. Rabbit antibodies are known for their high affinity, specificity and diversity, and furthermore rabbits and mice are evolutionarily diverse, so that rabbit antibodies are able to recognize human epitopes that are not immunogenic in mice, increasing the total number of targetable epitopes, helping to produce cross-reactive antibodies with human antigens. Anti-rabbit IgG antibodies are used in immunology as secondary antibodies for detection of rabbit primary antibodies, which can be conjugated to a label to help detect and quantify the binding of rabbit antibodies to specific antigens. Therefore, the preparation of anti-rabbit IgG antibodies with high sensitivity and accuracy is of great significance for scientific research, diagnostic detection and therapeutic applications. Sheep derived antibodies have unique advantages as secondary antibodies, sheep having 5 immunoglobulin isotypes, consisting of 5 heavy chain classes (IgM, igG, igA, igD and IgE), the IgG class being further subdivided into 3 subtypes (IgG 1, igG2, igG 3). As with other species, the heavy chain is associated with 2 light chains (Lambda and kappa). IgG-lambda accounted for 95% of IgG in sheep. Sheep antibodies typically have a 10 to 100 fold higher affinity than rodent antibodies, secondly, sheep monoclonal antibodies bind only to a specific region and aim to accurately distinguish closely related molecules (e.g. derivatives), potentially producing highly specific antibodies against the desired target, in addition to the unique immune system of sheep, sheep antibodies recognize "difficult" targets for which other antibody technologies have failed, and natural sheep antibodies recognize a variety of heterologous epitopes. In the current technical field, the goat anti-rabbit IgG antibody (Dylight 488) provided by GeneTex is a polyclonal antibody with low specificity, and the biotin-labeled goat anti-rabbit IgG (H+L) provided by Biyun is prepared by immunizing goat with rabbit IgG, purifying the obtained antiserum by using an affinity purification column, and obtaining the antiserum after adsorbing and purifying human IgG, mouse IgG and rat IgG, and is still a non-monoclonal antibody, so that the specificity of the anti-rabbit IgG is still improved. Disclosure of Invention 1. Object of the invention The application aims to provide a goat recombinant monoclonal antibody of anti-rabbit IgG, and preparation and application thereof, wherein a novel goat recombinant monoclonal antibody of anti-rabbit IgG is successfully prepared by a single B cell PCR technology, can specifically identify rabbit IgG proteins in tissues, can be singly or after being marked by horseradish peroxidase (HRP) and applied to detection of rabbit IgG proteins by means of immunohistochemistry, (indirect/direct) ELISA, protein immunoblotting and the like, has higher binding affinity with the rabbit IgG, so that the capability of detecting the rabbit IgG is remarkably improved, and provides a more reliable tool for accurate rabbit antibody detection. 2. Technical proposal In order to achieve the above purpose, the technical scheme adopted by the application is as follows: The application provides a goat recombinant monoclonal antibody of anti-rabbit IgG, which can be specifically combined with rabbit IgG protein, wherein the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region is shown as SEQ ID NO. 2. Furthermore, the amino acid sequence of the heavy chain constant region of the goat recombinant monoclonal antibody of the anti-rabbit IgG is shown as SEQ ID NO.5, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 6. The application also provides a nucleic acid which codes for the heavy chain variable region and/or the light chain variable region of the goat recombinant monoclonal antibody of the anti-rabbit IgG. Further, the nucleic acid also encodes the heavy chain constant region and/or the light chain constant region of the goat recombinant monoclonal antibody of the anti-rabbit IgG. Further, the nucleotide sequence of the goat recombinant monoclonal antibody for en